Peptides for activation and inhibition of δPKC

ABSTRACT

Peptides able to inhibit or activate the translocation or function of δPKC are identified. Administration of the peptides for protection or enhancement of cell damage due to ischemia is described. Therapeutic methods to reduce damage to cells or to enhance damage to cells due to ischemia are also described, as well as methods for screening test compounds for δPKC-selective agonists and antagonists.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/838,732, filed on Aug. 14, 2007, which is now U.S. Pat. No.7,915,219, which is a continuation of U.S. application Ser. No.10/843,731, filed on May 12, 2004, which is now U.S. Pat. No. 7,745,388,which is a divisional of U.S. application Ser. No. 10/007,761, filed onNov. 9, 2001, which is now U.S. Pat. No. 6,855,693, which claims thebenefit of priority of U.S. Provisional Application No. 60/262,060,filed Jan. 18, 2001, now expired, all of which are incorporated hereinby reference in their entirety.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This invention was made with Government support under contract HL052141awarded by the National Institutes of Health. The Government has certainrights in this invention.

REFERENCE TO SEQUENCE

A Sequence Listing is being submitted electronically via EFS in the formof a text file, created Nov. 30, 2010, and named586008208US04seqlist.txt (17521 bytes), the contents of which areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to peptides effective to activate orinhibit translocation and/or function of δPKC. The present inventionalso relates to therapeutic compositions and methods for treatingdiseases or conditions which are benefited by inhibition or activationof δPKC.

BACKGROUND OF INVENTION

Protein kinase C (PKC) is a key enzyme in signal transduction involvedin a variety of cellular functions, including cell growth, regulation ofgene expression and ion channel activity. The PKC family of isozymesincludes at least 11 different protein kinases which can be divided intoat least three subfamilies based on their homology and sensitivity toactivators. Members of the classical or cPKC subfamily, α, β_(I), β_(II)and γPKC, contain four homologous domains C1, C2, C3 and C4)inter-spaced with isozyme-unique (variable or V) regions, and requirecalcium, phosphatidylserine (PS), and diacylglycerol (DG) or phorbolesters for activation. Members of the novel or nPKC subfamily, (δ, ε, η,and θPKC, lack the C2 homologous domain and do not require calcium foractivation. Finally, members of the atypical or αPKC subfamily, ζ andλ/τPKC, lack both the C2 and one half of the C1 homologous domains andare insensitive to DG, phorbol esters and calcium.

Studies on the subcellular distribution of PKC isozymes demonstrate thatactivation of PKC results in its redistribution in the cells (alsotermed translocation), such that activated PKC isozymes associate withthe plasma membrane, cytoskeletal elements, nuclei, and othersubcellular compartments (Saito, et al., Proc. Natl. Sci. USA86:3409-3413 (1989); Papadopoulos, V. and Hall, P. F. J. Cell Biol.108:553-567 (1989); Mochly-Rosen, D., et. al., Molec. Biol. Cell(formerly Cell Reg.) 1:693-706 (1990)).

It appears that the unique cellular functions of different PKC isozymesare determined by their subcellular location. For example, activatedβ_(I)PKC is found inside the nucleus, whereas activated β_(II)PKC isfound at the perinueleus and cell periphery of cardiac myocytes(Disatnik, M. H., et al., Exp. Cell Res. 210:287-297 (1994). Further, inthe same cells, εPKC binds to cross-striated structures (possibly thecontractile elements) and cell-cell contacts following activation orafter addition of exogenous activated εPKC to fixed cells (Mochly-Rosen,al., 1990; Disatnik, et al., 1994). The localization of different PKCisozymes to different areas of the cell in turn appears due to bindingof the activated isozymes to specific anchoring molecules termedReceptors for Activated C-Kinase (RACKS).

RACKs are thought to function by selectively anchoring activated PKCisozymes to their respective subcellular sites. RACKs bind only fullyactivated PKC and are not necessarily substrates of the enzyme. Nor isthe binding to RACKs mediated via the catalytic domain of the kinase(Mochly-Rosen, D., et al., Proc. Natl. Acad. Sci. USA 88:3997-4000(1991)). Translocation of PKC reflects binding of the activated enzymeto RACKs anchored to the cell particulate fraction and the binding toRACKs is required for PKC to produce its cellular responses(Modify-Rosen, D., al. Science 268:247-251 (1995)). Inhibition of PKCbinding to RACKs in vivo inhibits PKC translocation and PKC-mediatedfunction (Johnson, J. A., Biol. Chem. 271:24962-24966 (1996a); Ron, D.,et al., Proc. Natl. Acad. Sci. USA 92:492-496 (1995); Smith, B. L. andMochly-Rosen, D., Biochem. Biophys. Res. Commun. 188:1235-1240 (1992)).

cDNA clones encoding RACK1 and RACK2 have been identified (U.S. Pat. No.5,519,003; Ron, D., et al., Proc. Natl. Acad. Sci. USA 91:839-84-3(1994); Csukai, M., et al., 9^(TH) INTERNATIONAL CONFERENCE ON SECONDMESSENGERS AND PHOSPHOPROTEINS 112 (1995)). Both are homologs of the βsubunit of G proteins, a receptor for another translocating proteinkinase, the β-adrenergic receptor kinase, βARK (Pitcher, J., et al.,Science 257:1264-1267 (1992)). Similar to Gβ, RACK1, and RACK2 haveseven WD40 repeats (Ron, et al., 1994; Csukai, et al., 1995). Recentdata suggest that RACK1 is a β_(II)PKC-specific RACK (Stebbins, E. G.,et al., Biol. Chem., 276:29644-29650 (2001)) and that RACK2 (Csukai, M.,et al, J. Biol. Chem., 272:29200-29206 (1997)) is specific for activatedεPKC.

Translocation PKC is required for proper function of PKC isozymes.Peptides that mimic either the PKC-binding site on RACKs (Mochly-Rosen,D., et al., J. Biol. Chem., 226:1466-1468 (1991a); Mochly-Rosen et 1995)or the RACK-binding site on PKC (Ron, et al., 1995; Johnson, et al.,1996a) are isozyme-specific translocation inhibitors of PKC thatselectively inhibit the function of the enzyme in vivo. For example, aneight amino acid peptide derived from εPKC (peptide εV1-2; SEQ ID NO:1,Glu Ala Val Ser Leu Lys Pro Thr) is described in U.S. Pat. No.6,165,977. The peptide contains a part of the RACK-binding site on εPKCand selectively inhibits specific εPKC-mediated functions in cardiacmyocytes. This εPKC peptide has been shown to be involved in cardiacpreconditioning to provide protection from ischemic injury. Prolongedischemia causes irreversible myocardium damage primarily due to death ofcells at the infarct site. Studies in animal models, isolated heartpreparations and isolated cardiac myocytes in culture have demonstratedthat short bouts of ischemia of cardiac muscle reduce such tissue damagein subsequent prolonged ischemia (Liu, Y., et al., J. Mol. Cell.Cardiol. 27:883-892 (1995), 1996; Hu, K. and Nattel, S., Circulation92:2259-2265 (1995); Brew, E. C., et al., Am. J. Physiol 269 (HeartCirc. Physiol, 38):H1370-H1378 (1995); Schultz, J. E. J., et al., Circ.Res. 78:1100-1104 (1996). This protection, which occurs naturallyfollowing angina and has been termed preconditioning, can be mimicked bya variety of non-specific PKC agonists (Mitchell, M. B., et al.,Circulation 88:1633 (1993); Mitchell, M. B., et al., Circ. Res. 76:73-81(1995); Murry, C. F., et al., Circulation 74:1123-1136 (1986);Speechly-Dick, M. E., et al., Circ. Res. 75:586-590 (1993)). Both δPKCand εPKC activation occurs following preconditioning (Gray, M. O. etal., J. Biol. Chem., 272:30945-3095 (1997)), however, εPKC activation isrequired for protection of cardiac myocytes from ischemia-induced celldeath (U.S. Pat. No. 6,165,977).

In a recent study, an εPKC-selective peptide agonist was shown toprovide cardioprotection from ischemia when administered intracellularto isolated neonatal and adult cardiomyocytes and when producedintracellularly in vivo in transgenic mice (Dorn, G., Proc. Natl. Acad.Sci. USA 96(22):12798-12803 (1999)).

The ability of δPKC peptide agonists and antagonists to protect cellsand tissue from on ischemic event or to reverse or reduce damage causedby an ischemic event has not been reported. More particularly, it isunknown in the art whether or not δPKC peptide agonists and antagonistscan be delivered extracellularly to whole tissue or intact organs invivo to achieve a therapeutic effect.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the invention to provide a method ofprotecting tissue from damage due to an ischemic event.

It is a further object of the invention to provide a method ofadministering an δPKC peptide antagonist for protection of cells andtissue from damage due to an ischemic event.

It is yet another object of the invention to provide a method ofameliorating damage to tissue caused by an ischemic event.

It is still a further objective of the invention to provide a method ofreducing or protecting cells and tissue from damage as a result ofstroke.

It is another objective of the invention to provide a method ofenhancing cellular or tissue damage as a result of an ischemic orhypoxic event.

In one aspect, the invention includes a peptide selected δV1-1 (SEQ IDNO:4), δV1-2 (SEQ ID NO:5), ψδRACK (SEQ ID NO:6), δV1-5 (SEQ ID NO:7),and derivatives and fragments thereof. Exemplary derivatives of δV1-1are identified as SEQ ID NOS:34-48. Exemplary derivatives of δV1-2 areidentified as SEQ ID NOS:65-71. Exemplary derivatives of ψδRACK areidentified as SEQ ID NOS:11-19, 22-33. Exemplary fragments of δV1-1 areidentified as SEQ ID NOS:49-64. Exemplary fragments of ψδRACK areidentified as SEQ ID NO:20 and SEQ ID NO:21.

In one embodiment, the peptide is recombinantly produced, such as wherethe peptide is encoded by a polynucleotide. In other embodiments, thepeptide is chemically synthesized.

In one embodiment, the peptide is linked to a moiety effective tofacilitate transport across a cell membrane. Exemplary moieties includea Tat-derived peptide, an Antennapedia carrier peptide, and apolyarginine peptide.

In another embodiment, the peptide is joined to a second peptide to forma fusion peptide.

In another aspect, the invention includes a method of reducing ischemicinjury to a cell or a tissue exposed to hypoxic conditions byadministering to the cell or tissue an amount of an isozyme-specificδPKC antagonist. Contemplated antagonists include δV1-1 (SEQ ID NO:4),δV1-2 (SEQ ID NO:5), δV1-5 (SEQ ID NO:7), and derivatives and fragmentsthereof.

In various embodiments of this method, the peptide is administered priorto, during or after exposing the cell or tissue to said hypoxicconditions. The peptide can be linked to a carrier peptide, as describedabove.

In one embodiment, the peptide is administered by infusion throughcoronary arteries to an intact heart.

In another aspect, the invention includes a method of reducing orpreventing or ameliorating damage to a cell or tissue due to stroke byadministering to the cell or tissue an amount of an isozyme-specificδPKC antagonist. Contemplated antagonists include δV1-1 (SEQ ID NO:4),δV1-2 (SEQ ID NO:5), δV1-5 (SEQ ID NO:7), and derivatives and fragmentsthereof.

In various embodiments of this method, the peptide is administered priorto, during or after the stoke, when the cell or tissue is exposed to ahypoxic event. The peptide can be linked to a carrier peptide, asdescribed above.

In another aspect, the invention includes a method of enhancing damageto a cell exposed to hypoxic conditions by administering to the cell anamount of an isozyme-specific specific δPKC agonist. Contemplatedagonists include ψδRACK identified as SEQ ID NO:6, derivatives andfragments or ψδRACK. Exemplary derivatives include peptides identifiedas SEQ ID NOS:11-19, and SEQ ID NOS:22-29. Exemplary fragments includethe peptides identified as SEQ ID NOS:20-21.

In one embodiment, the peptide is administered to a tumor cell. Theagonist peptide can be linked to a moiety effective to facilitatetransport across a cell membrane.

In another aspect, the invention includes a method of identifying acompound effective to induce protection of a cell from hypoxic orischemic damage. In the method, a δPKC peptide containing a δRACKbinding site is contacted with a δPKC antagonist δPKC peptide with theδRACK binding site in the presence and absence of said test compound.The test compound is identified as being effective to induce protectionif (i) binding in the presence of the test compound is decreasedrelative to binding in the absence of the test compound, or (ii)catalytic activity of the test compound is increased relative toactivity in the absence of the test compound.

In this method, the δPKC peptide can be selected from the groupconsisting of δV1-1 (SEQ ID NO:4), δV1-2 (SEQ ID NO:5), δV1-5 (SEQ EDNO:7), and fragments and derivatives thereof.

In another aspect, the invention includes a method of identifying acompound effective to enhance hypoxic or ischemic damage in a cell. AψδRACK agonist peptide is contacted with a δPKC peptide containing aRACK binding site in the presence and absence of a test compound. Thetest compound is identified as being effective to enhance ischemicdamage if (i) binding in the presence of the test compound is decreasedrelative to binding in the absence of the test compound, or (ii) thecatalytic activity of the δPKC in the presence of the test compound isincreased relative to the catalytic activity in the absence of the testcompound.

In one embodiment, a ψδRACK peptide selected from the group consistingof SEQ ID NO:6, fragments, and derivatives thereof is used. Exemplarysuitable derivatives and fragments are identified SEQ ID NOS:11-29.

In another aspect, the invention includes a method of providingprotection to tissue from damage caused by an ischemic or hypoxic eventby administering to the tissue a peptide selected from the groupconsisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, derivatives andfragments thereof. Suitable derivatives and fragments include thosegiven above.

In one embodiment, the peptide is administered by the intravenous,parenteral, subcutaneous, inhalation, intranasal, sublingual, mucosal,and transdermal route. In another method, the peptide is administeredduring a period of reperfusion; that is, after a period of initialperfusion.

Protection against ischemia is provided to a variety of tissues,including but not limited to the brain, heart, eye, and kidney.

These and other objects and features of the invention will be more fullyappreciated when the following detailed description of the invention isread in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the alignment of the primary sequence of rat δPKC (SEQ IDNO:2 from residue 1 to 123) and mouse ΘPKC, V1 (SEQ ID NO:3 from residue1 to 124) domains. The bracketed areas designated as δV1-1 (SEQ IDNO:4), δV1-2 (SEQ ID NO:5), and ψδR (SEQ ID NO:6) indicate regions ofdifference between the two isozymes.

FIG. 2A shows a Western blot autoradiogram of soluble (S) andparticulate (P) cell fractions after treatment with δV1-1in the presenceand absence of phorbol 2-myristate 13-acetate (PMA) and probing withanti-δPKC and anti-εPKC antibodies.

FIG. 2B shows the translocation of δPKC and εPKC, expressed as theamount of isozyme in the particulate fraction over the amount of isozymein non-treated cells, for cells treated as indicated in FIG. 2A withδV1-1 in the presence (+) and absence (−) of PMA.

FIG. 3A shows a Western blot autoradiogram of soluble (S) andparticulate (P) cell fractions after treatment with ψδRACK or with PMAand probing with anti-δPKC and anti-αPKC antibodies.

FIG. 3B shows the translocation of δPKC and αPKC, expressed as theamount of isozyme in the particulate fraction over the amount of isozymein non-treated cells, for cells treated as indicated in FIG. 3A withψδRACK in the presence (+) and absence (−) of PMA.

FIG. 4A shows a Western blot autoradiogram of soluble (S) andparticulate (P) cell fractions after treatment with δV-1 in the presenceand absence of ψδRACK and probing with anti-δPKC and anti-εPKCantibodies.

FIG. 4B shows the translocation of δPKC, expressed as the amount ofisozyme in the particulate fraction over the amount of isozyme innon-treated cells, for cells treated as indicated in FIG. 4A with δV1-1in the presence (+) and absence (−) of ψδRACK.

FIG. 5A shows percentage of cell damage for isolated cardiac myocytestreated with δV1-1 in the absence (−) or presence (in the concentrationsindicated along the x-axis) of ψδRACK. The peptides were administered 10minutes prior to a 180 minute ischemic period. As a control,δPKC-selective activator peptide was used.

FIG. 5B shows percentage of cell damage for isolated cardiac myocytestreated with δV1-1 in the absence (−) or presence (in the concentrationsindicated along the x-axis). The peptides were administered 10 minutesprior to a 90 minute ischemic period.

FIG. 6A shows the cell damage, as measured by creatine phosphokinase(CPK) release in whole rat hearts treated ex vivo with δV1-1 (solidcircles) or with ψδRACK (solid diamonds) as a function of timepost-ischemia and post-treatment. As controls, some hearts were leftuntreated prior to ischemia (open squares) and other hearts weremaintained in normoxia conditions (open triangles).

FIG. 6B is a bar graph showing the total cell damage, as measured bytotal CPK release for the ex vivo hearts treated as described in FIG. 6Awith δV1-1 and with ψδRACK, as well as ex vivo hearts treated with twocontrols: the Tat-carrier peptide alone and with a scrambled δV1-1sequence conjugated to Tat-carrier peptide.

FIGS. 7A-7B show the functional recovery of a working heart perfusedwith δV1-1 (FIG. 7A) or left untreated (FIG. 7B) after 20 minutes ofglobal ischemia, where the left ventricle developed pressure (LVP, inmmHg), its first derivative (dP/dt, in mmHg/sec), and the coronaryperfusion pressure (PP, in mmHg) are shown. On the right, an expandedtrace of the same functional measurement are shown before (base line)and 30 minutes after reperfusion.

FIGS. 8A-8C are plots of percent of left ventricular developed pressure(% LVDP, FIG. 8A), end diastolic pressure (EDP, FIG. 8B) and perfusionpressure (PP, FIG. 8C) of a working heart as a function of time beforeischemia (baseline; and 5 to 30 minutes after ischemia and duringtreatment δV1-1 (closed squares or untreated (open circles).

FIGS. 9A-9B are photos obtained by a digital camera of pig heart slicestaken from the pigs five days after treatment in vivo with δV1-1 (FIG.9A) or with the carrier peptide alone as a control (FIG. 9B) during thelast 10 minutes of a 30 minute ischemic insult.

FIG. 9C is a bar graph showing the percent of infarct of the area atrisk determined from the heart slices of FIGS. 9A-9B, for the pigstreated with δV1-1 and for the untreated, control animals.

FIG. 10 is a graph showing the ejection fraction, as measured by leftventricurogram in pigs at three time points; (1) before occlusion ofleft anterior descending artery by balloon catheter (pre ischemia); (2)immediately after reperfusion with δV1-1 (post ischemia); and (3) beforesacrifice five days later (5 days post ischemia), for animals treatedwith δV1-1 (solid circles) and for control animals treated with ascrambled peptide (open circles).

FIGS. 11A-11B are digitized photographs of brains taken from untreatedanimals (FIG. 11A) and animals treated with (δV1-1 (FIG. 11B) prior toan induced stroke.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is an eight amino acid peptide derived from εPKC, referredto as εV1-2 and described in U.S. Pat. No. 6,165,977.

SEQ ID NO:2 corresponds to amino acids 1-141 from the V1 domain of ratδPKC (accession no. KIRTCD).

SEQ ID NO:3 corresponds to amino acids 1-124 of V1 domain of mouse θPKC(accession no, Q0211.1).

SEQ ID NO:4 is an amino acid sequence from the first variable region ofδPKC (amino acids 8-17), δV1-1.

SEQ ID NO:5 is an amino acid sequence from the first variable region ofδPKC (amino acids 35-45), δV1-2.

SEQ ID NO:6 is an amino acid sequence from δPKC, (amino acids 74-81),and is referred to herein as “pseudo-delta” RACK, or ψδRACK.

SEQ ID NO:7 is an amino acid sequence from a region of δPKC (amino acids619-676), referred to herein as δV1-5.

SEQ ID NO:8 is the Drosophila Antennapedia homeodomain-derived carrierpeptide.

SEQ ID NO:9 is a Tat-derived carrier peptide (Tat 47-57),

SEQ ID NO:10 is a βPKC-selective activator peptide.

SEQ ID NO:11 is a modification of SEQ ID NO:6 (ψδRACK),

SEQ ID NO:12 is a modification of SEQ ID NO:6 (ψδRACK).

SEQ ID NO:13 is a modification of SEQ ID NO:6 (ψδRACK).

SEQ ID NO:14 is a modification of SEQ ID NO:6 (ψδRACK).

SEQ ID NO:15 is a modification of SEQ ID NO:6 (ψδRACK).

SEQ ID NO:16 is a modification of SEQ ID NO:6 (ψδRACK).

SEQ ID NO:17 is a modification of SEQ ID NO:6 (ψδRACK).

SEQ ID NO:18 is a modification of SEQ ID NO:6 (ψδRACK).

SEQ ID NO:19 is a modification of SEQ ID NO:6 (ψδRACK).

SEQ ID NO:20 is a fragment of SEQ ID NO:6 (ψδRACK).

SEQ ID NO:21 is a fragment of SEQ ID NO:6 (ψδRACK).

SEQ ID NO:22 is a modification of SEQ ID NO:6 (ψδRACK).

SEQ ID NO:23 is a modification of SEQ ID NO:6 (ψδRACK).

SEQ ID NO:24 is a modification of SEQ ID NO:6 (ψδRACK).

SEQ ID NO:25 is a modification of SEQ ID NO:6 (ψδRACK).

SEQ ID NO:26 is a modification of SEQ ID NO:6 (ψδRACK).

SEQ ID NO:27 is a modification of SEQ ID NO:6 (ψδRACK).

SEQ ID NO:28 is a modification of SEQ ID NO:6 (ψδRACK).

SEQ ID NO:29 is a modification of SEQ ID NO:6 (ψδRACK).

SEQ ID NO:30 is a modification of SEQ ID NO:6 (ψδRACK).

SEQ ID NO:31 is a modification of SEQ ID NO:6 (ψδRACK).

SEQ ID NO:32 is a modification of SEQ ID NO:6 (ψδRACK).

SEQ ID NO:33 is a modification of SEQ ID NO:6 (ψδRACK).

SEQ ID NO:34 is a modification of SEQ ID NO:4 (δV1-1).

SEQ ID NO:35 is a modification of SEQ ID NO:4 (δV1-1).

SEQ ID NO:36 is a modification of SEQ ID NO:4 (δV1-1).

SEQ ID NO:37 is a modification of SEQ ID NO:4 (δV1-1).

SEQ ID NO:38 is a modification of SEQ ID NO:4 (δV1-1).

SEQ ID NO:39 is a modification of SEQ ID NO:4 (δV1-1).

SEQ ID NO:40 is a modification of SEQ ID NO:4 (δV1-1).

SEQ ID NO:41 is a modification of SEQ ID NO:4 (δV1-1).

SEQ ID NO:42 is a modification of SEQ ID NO:4 (δV1-1).

SEQ ID NO:43 is a modification of SEQ ID NO:4 (δV1-1).

SEQ ID NO:44 is a modification of SEQ ID NO:4 (δV1-1).

SEQ ID NO:45 is a modification of SEQ ID NO:4 (δV1-1).

SEQ ID NO:46 is a modification of SEQ ID NO:4 (δV1-1).

SEQ ID NO:47 is a modification of SEQ ID NO:4 (δV1-1).

SEQ ID NO:48 is a modification of SEQ ID NO:4 (δV1-1).

SEQ ID NO:49 is a fragment of SEQ ID NO:4 (δV1-1).

SEQ ID NO:50 is a modified fragment of SEQ ID NO:4 (δV1-1).

SEQ ID NO:51 is a modified fragment of SEQ ID NO:4 (δV1-1).

SEQ ID NO:52 is a modified fragment of SEQ ID NO:4 (δV1-1).

SEQ ID NO:53 is a modified fragment of SEQ ID NO:4 (δV1-1).

SEQ ID NO:54 is a modified fragment of SEQ ID NO:4 (δV1-1).

SEQ ID NO:55 is a modified fragment of SEQ ID NO:4 (δV1-1).

SEQ ID NO:56 is a modified fragment of SEQ ID NO:4 (δV1-1).

SEQ ID NO:57 is a modified fragment of SEQ ID NO:4 (δV1-1).

SEQ ID NO:58 is a fragment of δV1-1.

SEQ ID NO:59 is a fragment of δV1-1.

SEQ ID NO:60 is a fragment of δV1-1.

SEQ ID NO:61 is a fragment of δV1-1.

SEQ ID NO:62 is a fragment of δV1-1.

SEQ ID NO:63 is a fragment of δV1-1.

SEQ ID NO:64, is a fragment of δV1-1.

SEQ ID NO:65 is a modification of SEQ ID NO:5 (δV1-2).

SEQ ID NO:66 is a modification of SEQ ID NO:5 (δV1-2).

SEQ ID NO:67 is a modification of SEQ ID NO:5 (δV1-2).

SEQ ID NO:68 is a modification of SEQ ID NO:5 (δV1-2).

SEQ ID NO:69 is a modification of SEQ ID NO:5 (δV1-2).

SEQ ID NO:70 is a modification of SEQ ID NO:5 (δV1-2).

SEQ ID NO:71 is a modification of SEQ ID NO:5 (δV1-2).

SEQ ID NO:72 is the sequence of Annexin V.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Unless otherwise indicated, all terms herein have the same meaning asthey would to one skilled in the art of the present invention.Practitioners are particularly directed to Current Protocols inMolecular Biology (Ausubel, F. M. et al., John Wiley and Sons, Inc.,Media Pa.) for definitions and terms of the art.

Abbreviations for amino acid residues are the standard 3-letter and/or1-letter codes used in the art to refer to one of the 20 common L-aminoacids.

A “conserved set” of amino acids refers to a contiguous sequence ofamino acids that is conserved between members of a group of proteins. Aconserved set may be anywhere from two to over 50 amino acid residues inlength. Typically, a conserved set is between two and ten contiguousresidues in length. For example, for the two peptides MKAAEDPM (SEQ IDNO:11) and MRAPEDPM (SEQ ID NO:14), there are 4 identical so positions(EDPM; SEQ ID NO:20) which form the conserved set of amino acids forthese two sequences.

“Conservative amino acid substitutions” are substitutions which do notresult in a significant change in the activity (e.g., δV1-1 PKCactivity) or tertiary structure of a selected polypeptide or protein.Such substitutions typically involve replacing a selected amino acidresidue with a different residue having similar physico-chemicalproperties. For example, substitution of Glu for Asp is considered aconservative substitution since both are similarly-sizednegatively-charged amino acids. Groupings of amino acids byphysico-chemical properties are known to those of skill in the art.

“Peptide” and “polypeptide” are used interchangeably herein and refer toa compound made up of a chain of amino acid residues linked by peptidebonds. Unless otherwise indicated, the sequence for peptides is given inthe order from the amino terminus to the carboxyl terminus.

Two amino acid sequences or two nucleotide sequences are consideredhomologous (as this term is preferably used in this specification) ifthey have an alignment score of >5 (in standard deviation units) usingthe program ALIGN with the mutation gap matrix and a gap penalty of 6 orgreater (Dayhoff, M. O., in ATLAS OF PROTEIN SEQUENCE AND STRUCTURE(1972) Vol. 5, National Biomedical Research Foundation, pp. 101-110, andSupplement 2 to this volume, pp. 1-10.) The two sequences (or partsthereof) are more preferably homologous if their amino acids are greaterthan or equal to 50%, more preferably 70%, still more preferably 80%,identical when optimally aligned using the ALIGN program mentionedabove.

A peptide or peptide fragment is “derived from” a parent peptide orpolypeptide if it has an amino acid sequence that is identical orhomologous to the amino acid sequence of the parent peptide orpolypeptide.

“Ischemia” or an “ischemic event” refers to an insufficient supply ofblood to a specific cell, tissue or organ. A consequence of decreasedblood supply is an inadequate supply of oxygen to the organ or tissue(hypoxia). Prolonged hypoxia may result in injury to the affected organor tissue.

“Anoxia” refers to a virtually complete absence of oxygen in the organor tissue, which, if prolonged, may result in death of the cell, organor tissue.

“Hypoxia” or a “hypoxic condition” intend a condition under which acell, organ or tissue receive an inadequate supply of oxygen.

“Reperfusion” refers to return of fluid flow into a tissue after aperiod of no-flow or reduced flow. For example, in reperfusion of theheart, fluid or blood returns to the heart through the coronary arteriesafter occlusion of these arteries has been removed.

“Tissue” as used herein intends a whole organ, either in vivo or exvivo, a fragment of an organ, or two or more cells.

The term “PKC” refers to protein kinase C, or C-kinase.

The term “RACK” refers to receptor for activated C-kinase.

II. δPKC Peptide Agonists and Antagonists

In one aspect, the invention includes peptides effective to activateδPKC and peptides effective to inhibit δPKC. The sequence of the RACKfor δPKC is unknown as this RACK has not yet been identified. Thus, itis a challenge to identify δPKC-selective activator and inhibitorpeptides in the absence of any information about the δRACK sequence.Further, the exact role of δPKC in response to ischemia is also notknown in the art. It is known that δPKC, like εPKC, undergoestranslocation ischemic preconditioning in rat (Gray, M. O. et al, J.Biol. Chem., 272:30945-3095 (1997); Chen, C.-H. et al., Proc. Natl.Acad. Sci. USA, 96:12784-12789 (1999)). However, whether the δPKCtranslocation results in protection from ischemia or not has beenunknown until the present invention.

In studies performed in support of the present invention to identifypeptide sequences for activation and inhibition of δPKC, the sequence ofδPKC was compared to the sequence of θPKC, since of the three othernovel PKC isozymes, δPKC is most similar to θPKC with a 52% identity ofamino acid sequence (Osada, S.-I, et al., Molec. Cell. Biol.,12:3930-393 (1992); Baier, G. et al. J. Biol. Chem., 268:4997-5004(1993)). It was also assumed that each PKC isozyme should interact witha different RACK. Since the first variable (V1) domain of δPKC containsthe RACK-binding site (Johnson et al. 1996a) regions least similar toθPKC may be involved in RACK binding. FIG. 1 compares the sequences ofthe V1 domain of rat δPKC (SEQ ID NO:2; Accession No. KIRTCD) and MouseθPKC V1 domain (SEQ ID NO:3, accession no. Q02111). Three regions in theV1 domain of θPKC were identified with only ˜10% identity to θPKC. Theseregions are indicated in FIG. 1 by the bars above the sequence of δPKCand are referred to herein as δV1-1 having a sequence identified hereinas SEQ ID NO:4 (SFNSYELGSL), δV1-2 having a sequence identified hereinas SEQ ID NO:5 (ALTTDRGKTLV), and ψδRACK having a sequence identifiedherein as SEQ ID NO:6 (MRAAEDPM). Not shown in FIG. 1 is yet anothersequence identified from the δPKC sequence for testing of its activationor inhibition of δPKC. This sequences is identified as SEQ ID NO:7 andis referred to herein as δV1-5.

As described in Example 1, the δV1-1 and ψδRACK peptides were analyzedto determine whether the peptides had activity, and if so, whether theactivity was as an agonist or an antagonist of δPKC. As will be shown,δV1-1, δV1-2 and δV1-5 are δPKC antagonists and ψδRACK is a δPKCagonist. In these studies, the δV1-1 and ψδRACK peptides were modifiedwith a carrier peptide by cross-linking via an N-terminal Cys-Cys bondto the Drosophila Antennapedia homeodomain (SEQ ID NO:8; Théodore, L.,et al. J. Neurosci. 15:7158 (1995); Johnson, J. A., et al., Circ. Res.79:1086 (1996b)). In other studies, not described here, the peptide wasmodified with Tat (SEQ ID NO:9) or with polyarginine (Mitchell et al.,J. Peptide Res., 56:318-325 (2000). Rothbard et al., Nature Med.,6:1253-1257 (2000)) and gave results similar to those described herein.Details of the study are set forth in Example 1. In brief, theAntennapedia-conjugated peptides were introduced to cardiac cells at aconcentration of 500 nM in the presence and absence of phorbol12-myristate 13-acetate (PMA) or in the presence of each other.Translocation δPKC isozyme was assessed by Western blot analysiscystosolic and particulate fractions of treated cells. Subcellularlocalization of δPKC: isozyme was assessed by immunofluorescence byprobing the blot with anti-δPKC, anti-αPKC, and anti-ε PKC antibodies.Translocation was expressed as the amount of isozyme in the particulatefraction over the amount of isozyme in non-treated cells. The resultsare shown in FIGS. 2-4.

FIGS. 2A-2B show the results for the cells treated with W in thepresence (+) and absence (−) of PMA, FIG. 2A is the Western blotautoradiogram of soluble (S) and particulate (P) cell fractions aftertreatment with the peptide and after probing with anti-δPKC andanti-εPKC antibodies. FIG. 2B shows the translocation of δPKC expressedas the amount of isozyme in the particulate fraction over the amount ofisozyme in non-treated cells. The δV1-1 peptide inhibited PMA-inducedδPKC translocation. In other studies not shown here, the δV1-1 peptidedid not inhibit the translocation of εPKC or αPKC.

FIGS. 3A-3B are similar plots for the cells treated with ψδRACK in thepresence (+) and absence (−) PMA. ψδRACK was opposite in effect fromδV1-1 in that it selectively induced δPKC translocation in cardiacmyocytes, without affecting the translocation of PKCα or εPKC (notshown).

FIGS. 4A-4B shows the results for the cells treated with δV1-1 in thepresence and absence of ψδRACK. Basal partitioning of δPKC in theparticulate fraction was inhibited by δV1-1 and the presence of ψδRACKreversed this δV1-1 effect.

Together the results in FIGS. 2-4 shows that δV1-1 is a selectivetranslocation inhibitor of δPKC and that ψδRACK is analogous to theψRACK site and acts as a selective translocation activator of δPKC.

A. Protection of Cells from Damage Due to Ischemia

In another study, the δPKC activator peptide, ψδRACK, and the δPKCinhibitor peptide, δV1-1 were administered to isolated rat cardiacmyocytes to determine the role of δPKC in protection from ischemia. Asdescribed in Example 2, the Antennapedia carrier-peptide conjugate ofδV1-1 and/or ψδRACK was introduced into isolated adult rat cardiacmyocytes ten minutes prior to prolonged ischemia. Cell damage wasassessed using an osmotic fragility test by measuring uptake of trypanblue. The results are shown in FIGS. 5A-5C.

FIG. 5A shows the results for cells treated with δV1-1 at concentrationsof 10 nM, 100 nM, 500 nM, and 1 μM in the presence or absence (−) of 1μM ψδRACK. The results are presented as the percentage of cell damagefor cells treated as indicated along the x-axis. As a control, aβPKC-selective activator peptide (SVEIWD, SEQ ID NO:10) was used. Thepeptides were administered ten minutes prior to the 180 minute ischemicperiod. The presence δV1-1 administered prior to ischemia resulted in aconcentration-dependent level of protection from ischemia-induceddamage. The protection was prevented by co-incubation with theδPKC-specific translocation activator peptide, ψδRACK, but not withco-incubation with the control βPKC-selective translocation activator.

The data in FIG. 5A suggested that activation of δPKC with ψδRACK causeda slight increase in cardiac myocyte damage after an ischemic insult.Based on this, ψδRACK was hypothesized as acting synergistically withischemia-induced activation of δPKC to cause cell damage. This wasevaluated by reducing the period of ischemic insult, since synergismbetween ψδRACK and ischemia in inducing cell damage should becomeapparent when ischemic insult was reduced. Thus, another study wasperformed where the ischemic period was shortened to 90 minutes. Theresults of this study are shown in FIG. 5B. The ψδRACK-induced increasein cell damage became significant when the time of ischemia wasshortened from 180 to 90 minutes, and was reversed by co-treatment withthe δPKC inhibitor, δV1-1. Therefore, activation of δPKC by ischemiaappears to mediate cell damage. Together, FIGS. 5A and 5B demonstratethat cell damage induced by simulated ischemia is due, at least in part,to activation of δPKC.

B. Ex Vivo Delivery of Peptides to Whole Hearts

In another study performed in support of the invention, the δPKCselective inhibitor peptide, δV1-1, or the activator peptide, ωδRACK,were delivered to whole hearts ex vivo to determine if the peptides haveactivity when introduced extracellularly to a whole organ. As describedin Example 3, δV1-1 and ψδRACK peptides were conjugated to a carrierpeptide, a Tat-derived peptide. The peptides were delivered intoLangendorff perfused rat hearts prior to induction of an ischemicperiod. After perfusion with the peptides, global ischemia was effectedfor 30 minutes. After the 30 minute ischemic period, the amount ofcreatine phosphokinase (CPK) released was monitored during a 30 minutereperfusion period. The results are shown in FIGS. 6A-6B.

FIG. 6A shows the cell damage, as measured by creatine phosphokinase(CPK) release in the whole rat hearts treated with δV1-1 (solid circles)or with ψδRACK solid diamonds) as a function of time during thepost-ischemia, reperfusion period. As controls, some hearts were leftuntreated prior to ischemia (open squares) and other hearts weremaintained in normoxia conditions (open triangles).

FIG. 6B is a bar graph showing the total cell damage, as measured bytotal CPK release for the ex vivo hearts treated as described in FIG. 6Awith δV1-1 and ψδRACK. FIG. 6B also shows the total cell damage for exvivo hearts treated with two controls: the Tat-carrier peptide alone andwith a scrambled ψδRACK sequence conjugated to Tat-carrier peptide.

FIGS. 6A-6B show that acute administration of the δPKC activator,ψδRACK, enhanced cardiac damage induced by ischemia by about 30%. Acuteadministration of the δPKC-selective inhibitor, δV1-1, protected heartsagainst ischemic damage as shown by decreased release of creatinekinase. Together, these data indicate that in an intact heart,inhibition of δPKC conferred greater than 50% protection againstischemic damage (FIG. 6A). Accordingly, the invention contemplates amethod of protecting a cell or a tissue from damage due to ischemia byadministering a δPKC-selective antagonist, such as δV1-1, δV1-2, δV1-5,to the tissue. Such administration is effective to reduce cell damage byat least about 10%, more preferably by at least about 25%, and mostpreferably by at least about 50% when compared to tissue left untreatedprior to an ischemic insult.

Another study was performed to determine if the peptides could bedelivered to an intact organ to provide protection after an ischemicinsult. In this study, as described in Example 4, the rat heart modeldescribed above was used and the hemodynamic parameters were measuredduring the 20 minutes of global ischemia and the 20 minutes ofreperfusion. During the reperfusion only, δV1-1 was delivered at aconcentration of 500 nM. The results are shown in FIGS. 7A-7B.

FIG. 7A shows the functional recovery of a working heart perfused withδV1-1 after 20 minutes of global ischemia, where the left ventricledeveloped pressure (LVP, in mmHg), its first derivative (dP/dt, inmmHg/see), and the coronary perfusion pressure (PP, in mmHg) are shown.FIG. 7B is a similar plot for an untreated heart. As seen by comparingthe traces for the δV1-1 treated heart (FIG. 7A) and the untreated heartFIG. 7B), when δV1-1 was delivered during the first 20 minutes ofreperfusion, there was a significant improvement in functional recoveryafter ischemia. In particular, a significant improvement in both the LVPrecovery and its first derivative (dP/dt) were achieved by administeringδV1-1 after ischemic insult. The δV1-1 peptide reduced the elevated LVPend diastolic pressure and the coronary perfusion pressure (PP). Inaddition there was a ˜50% reduction in creatine phosphokinase release ascompared with hearts treated with vehicle control (not shown).

In a similar study, five pairs of rats were treated as described inExample 4, where the ex vivo hearts were subjected to 20 minutes ofischemia and 30 minutes of reperfusion. During the first 20 minutes ofreperfusion, 500 nM of δV1-1 or vehicle control was administered. Theaveraged results are shown in FIGS. 8A-8C.

FIG. 8A shows the percent of left ventricular developed pressure (%LVDP) before ischemia, noted on the x-axis as “baseline” and during the5-30 minute period after reperfusion was provided. Data were collectedduring the reperfusion, meaning during and after treatment with δV1-1Hearts treated with the δV1-1 peptide (closed squares) had a 2-fold to4-fold higher LVDP than hearts left untreated (open circles).

FIG. 8B is a similar plot showing the end diastolic pressure (EDP)before ischemia, noted as “baseline” on the x-axis, and during the 5-30minute period after reperfusion treatment with δV1-1 (closed squares) orafter reperfusion with a control vehicle (open circles). The EDP forhearts treated with δV1-1 was approximately 60 mmHg. Hearts leftuntreated after ischemia (open circles) had an EDP of between about70-80 mmHg.

FIG. 8C shows the perfusion pressure (PP) of the δV1-1 treated hearts(closed squares) and the untreated hearts (open circles). The baselineperfusion pressure before ischemia is indicated on the x-axis. Afterischemia and after treatment with δV1-1 the perfusion pressure was about75% of that found hearts left untreated.

The data in FIGS. 7-8 show that administration of a δPKC antagonistpeptide, such as δV1-1, δV1-2, δV1-5, after an ischemic insult to a cellor tissue is effective to protect the cell or tissue from damage do toischemia and resulting hypoxia. The data also show that a δPKCantagonist peptide is effective to reduce or minimize the damage due toischemia and hypoxia allowing the tissue to recover its functionalproperties following ischemia.

C. In Vivo Treatment with δV1-1

In another study in support of the invention, the ability of δV1-1peptide to protect tissue from damage due to an ischemic or hypoxicevent was evaluated by administering the peptide in vivo to adult femalepigs. As detailed in Example 5, δV1-1 peptide was administered to thepigs during the last 10 minutes of a 30 minute ischemic insult. Fivedays later, the hearts were analyzed for tissue damage. The results areshown in FIGS. 9A-9B.

FIGS. 9A-9B are digitized photos of pig heart slices taken from the pigstreated in vivo five days earlier with δV1-1(FIG. 9A) or with thecarrier peptide alone as a control (FIG. 9B). The hearts were stainedwith a double-staining technique (Example 5) that allowed determinationof the area at risk for ischemic injury (area within the arrows, mainlyin the lower hemisphere between the two arrows) and infracted area(white area in FIG. 9B). As seen in FIG. 9B, control hearts have a largeinfarct area within the area at risk (borders shown with arrows). Incontrast, pigs that received the δV1-1 peptide (FIG. 9A) have asignificantly reduced infarct area. The white area in FIG. 9A that isoutside the area of risk (outside the arrows) is connective tissue andfat, and is not an infracted area.

FIG. 9C is a bar graph showing the percent of infarct of the area atrisk for the untreated, control animals and the animals treated withδV1-1. Animals treated with a δPKC antagonist had a nearly two-foldlower percentage of infarct than animals left untreated. Together, FIGS.9A-9C show that δV1-1 can be administered in vivo to a whole organ andprovide protection from damage due to ischemia.

Blood samples and tissue samples of lung, liver, brain, gut, kidney,etc. were collected from the animals and analyzed at a pathology lab.All samples were normal and no inflammation or tissue abnormalities wereobserved. In addition, there was no adverse effect of two injections ofthe δV1-1 antagonist peptide at 1 μM final concentration in the mousemodel. Kidney, liver, brain, and lung functions were normal and allblood analyses were also normal.

In another study, left ventricurogram was performed in pigs (n=5) atthree time points: (1) before occlusion of left anterior descendingartery by balloon catheter (pre ischemia); (2) immediately afterreperfusion with 2.5 μM/10 mL of δV1-1 (post ischemia); and (3) beforesacrifice five days later (5 days post ischemia), using 6 Fr. ofpig-tail catheter, LVG was recorded by 2 views, right anterior obliqueand left anterior oblique. Ejection fraction (EF), the percent of bloodejected in a beat, during maximum contraction, of the total maximumpresent in the left ventricle, was analyzed by the software, Plus Plus(Sanders Data Systems), and the averages of two views were evaluated.Ejection fractions were calculated based on left ventricle dimensionsand the results are shown in FIG. 10. Ejection fraction is a measure ofhow well the heart is functioning, with a higher ejection fractionindicative of a better functioning heart. An ejection fraction of lessthan 50% in a short period of time can suggest progression into a stateof heart failure. Animals treated with δV1-1 (solid circles) exhibited aless pronounced decrease in ejection fraction than did the controlanimals treated with a scrambled peptide (open circles), suggesting thatthe peptide is effective to reduce or prevent damage to the cells andtissue due to ischemia. This is also evident from the data point at livedays post ischemia., where animals treated with δV1-1 had an ejectionfraction on par with that measured prior to ischemia and significantlyhigher than the untreated animals.

In summary, the ex vivo and in vivo studies show that δV1-1, whendelivered before, during, or after ischemia, confers a substantialreduction of damage to the heart and brain induced by ischemia.Therefore, treatment with a δPKC peptide antagonist, such as δV1-δV1-2,δV1-5 peptides, provides a therapeutic treatment for tissues exposed toischemia, such as occurs during cardiac ischemia.

D. In Vivo Treatment for Inhibition of Stroke-Induce Damage

In another study performed in support of the invention, the ability ofδV1-1 peptide (SEQ ID NO:4) to inhibit damage to the brain as a resultof stroke was examined. In this study, described in Example 6, a ratcerebral ischemia model was used. Ischemia was induced using anintraluminal suture to occlude the ostium of the middle cerebral artery.δV1-1 conjugated to Tat peptide (SEQ ID NO:9) or the Tat peptide alonewere injected into the carotid artery before and after a two hourocclusion period. The brain from each animal was harvested 24 hourslater, stained, and examined. The results are shown in FIGS. 11A-11B.

FIG. 11A is a digitized photograph of brains taken from untreatedanimals subjected to an induced stroke. The stained rat brain sectionsclearly demonstrated a middle cerebral artery territory infarct. Theinfarct area induced by the two hours of occlusion was reproduciblebetween animals. FIG. 11B shows the brain sections from two animalstreated with δV1-1 peptide prior to ischemia and at the end of theischemic period. The significant reduction in infarct area is readilyapparent.

Accordingly, the invention contemplates a method of reducing damage totissue in the central nervous system, such as the brain, neurons, andglial cells, by administering a δPKC peptide antagonist, such as δV1-1,δV1-2, δV1-5, prior to, during, or after a stroke. The peptide iseffective to reduce the tissue damage, as evidenced by at least about a10% reduction in infarct area., more preferably at least about a 25%reduction, and most preferably, at least about a 50% reduction ininfarct area, when compared to untreated tissue exposed to the ischemicinsult.

III. Method of Use

As described above, the peptides of the invention, δV1-1, δV1-2, δV1-5,and ψδRACK, act as translocation inhibitors or activators of δPKC.ψδRACK is an agonist, inducing translocation of δPKC to promote celldamage due to ischemia and/or hypoxia. δV1-1, 6V1-2, and δV1-5 areantagonists, inhibiting δPKC translocation to prevent cell damage due toischemia and resulting hypoxia.

It will be appreciated that the peptides can be used in native form ormodified by conjugation to a carrier, such as those described above.Alternatively, one or two amino acids from the sequences can besubstituted or deleted and exemplary modifications and derivatives andfragments for each peptide are given below.

For the ψδRACK peptide, identified as SEQ ID NO:6, potentialmodifications include the following changes shown in lower case:MkAAEDPM (SEQ ID NO:11), MRgAEDPM (SEQ ID NO:12), MRAgEDPM (SEQ IDNO:13), MRApEDPM (SEQ NO:14), MRAnEDPM (SEQ ID NO:15), MRAAdDPM (SEQ IDNO:16), MRAAEDPv (SEQ ID NO:17), MRAAEDPi (SEQ ID NO:18), MRAAEDPl (SEQID NO:19), and MRAAEDmp (SEQ ID NO:22), MeAAEDPM (SEQ ID NO:23),MdAAEDPM (SEQ ID NO:24), MRAAEePl (SEQ ID NO:25), MRAAEDPI (SEQ IDNO:26), MRAAEePi (SEQ ID NO:27), MRAAEePv (SEQ ID NO:28), MRAAEDPv (SEQID NO:29), and any combination of the above. The following modificationsto ψδRACK are also contemplated and are expected to convert the peptidefrom agonist to an antagonist: MRAAnDPM (SEQ ID NO:30), and MRAAqDPM(SEQ ID NO:31), MRAAEqPM (SEQ ID NO:32), MRAAEnPM (SEQ ID NO:33).Suitable fragments of ψδRACK are also contemplated, and SEQ ID NOS: 20,21 are exemplary.

Accordingly, the term “a δPKC agonist” as used herein intends a ψδRACKpeptide, which refers to SEQ ID NO:6 and to peptides having a sequencehomologous SEQ ID NO:6 and to peptides identified herein, but notlimited to, as SEQ ID NO:11-19 and SEQ ID NO:21-29. The term a δPKCagonist further refers to fragments of these ψδRACK peptides, asexemplified by SEQ ID NOS:20-21.

For δV1-1, potential modifications include the following changes shownin lower case: tFNSYELGSL: (SEQ ID NO:34), aFNSYELGSL (SEQ ID NO:35),SFNSYELGtL (SEQ ID NO:36), including any combination of these threesubstitutions, such as tFNSYELGtL (SEQ ID NO: 37). Other potentialmodifications include SyNSYELGSL (SEQ ID NO:38), SFNSfELGSL (SEQ IDNO:39), SFNSYdLGSL (SEQ ID NO:40), SFNSYELGSv (SEQ ID NO:41). Otherpotential modifications include changes of one or two L to I or V, suchas SFNSYEiGSv (SEQ ID NO:42), SFNSYEvGSi, (SEQ ID NO:43) SFNSYELGSv (SEQID NO:44), SFNSYELGSi (SEQ ID NO:45), SFNSYEiGSL (SEQ ID NO:46),SFNSYEvGSL (SEQ ID NO:47), aFNSYELGSL (SEQ ID NO:48), and anycombination of the above-described modifications. Fragments andmodification of fragments of δV1-1 are also contemplated, such as YELGSL(SEQ ID NO:49), YdLGSL (SEQ ID NO:50), fdLGSL (SEQ ID NO:51), YdiGSL(SEQ ID NO:52), YdvGSE (SEQ ID NO:53), YdLpsL (SEQ ID NO:54), YdLglL(SEQ ID NO:55), YdLGSi (SEQ ID NO:56), YdLGSv (SEQ ID NO:57), LGSL (SEQID NO:58), iGSL (SEQ ID NO:59), vGSL (SEQ ID NO:60), LpSL (SEQ IDNO:61), LGlL (SEQ ID NO:62), LGSi (SEQ ID NO:63), LGSv (SEQ ID NO:64).

Accordingly, the term “a δV1-1 peptide” as used herein refers to apeptide identified by SEQ ID NO:4 and to peptides homologous to SEQ IDNO:4, including but not limited to the peptides set forth in SEQ IDNOS:34-48, as well as fragments of any of these peptides that retainactivity, as exemplified by but not limited to SEQ ID NOS:49-64.

For δV1-2, potential modifications include the following changes shownin lower ease: ALsTDRGKTLV (SEQ ID NO:65), ALTsDRGKTLV (SEQ ID NO:66),ALTTDRGKsLV (SEQ ID NO:67), and any combination of these threesubstitutions, ALTTDRpKTLV (SEQ ID NO:68), ALTTDRGrTLV (SEQ ID NO:69),ALTTDkGKTLV (SEQ ID NO:70), ALTTDkGkTLV (SEQ ID NO:71), changes alone ortwo L to I, or V and changes of V to or L and any combination of theabove. In particular, L and V can be changed to V, L, I R and D, E canchange to N or Q.

Accordingly, the term “a δV1-2 peptide” as used herein refers to apeptide identified by SEQ ID NO:5 and to peptides homologous SEQ IDNO:5, including hut not limited to the peptides set forth in SEQ IDNOS:65-71, as well as fragments of any of these peptides that retainactivity.

For δV1-5 (SEQ ID NO: 7), potential modifications include those similarto the modifications described for δV1-2. The term “a δV1-5 peptide” asused herein refers to SEQ ID NO:7 and to peptides homologous to SEQ IDNO:7 as well as fragments thereof that retain activity.

Accordingly, the term “a δPKC antagonist” as used herein intends a δPKCpeptide, which refers to a δV1-1 peptide, a δV1-2 peptide or a δV1-5peptide.

In still other embodiments, the peptide can be part of a fusion proteinor a transport protein conjugate. Typically, to form a fusion protein,the peptide is bound to another peptide by a bond other than a Cys-Cysbond. An amide bond from the C-terminal of one peptide to the N-terminalof the other is exemplary of a bond in a fusion protein. The secondpeptide to which the δPKC agonist/antagonist peptide is bound can bevirtually any peptide selected for therapeutic purposes or for transportpurposes. For example, it may be desirable to link the δV1-1 peptide toa cytokine or other peptide that elicits a biological response.

Where the peptide is part of a conjugate, the peptide is typicallyconjugated to a carrier peptide, such as Tat-derived transportpolypeptide (Vives et al. J. Biol. Chem., 272:16010-16017 (1997)),polyarginine (Mitchell et al., 2000; Rothbard et al., 2000) orAntennapedia peptide by a Cys-Cys bond. See U.S. Pat. No. 5,804,604. Inanother general embodiment, the peptides can be introduced to a cell,tissue or whole organ using a carrier or encapsulant, such as a liposomein liposome-mediated delivery.

The peptide may be (i) chemically synthesized or (ii) recombinantlyproduced in a host cell using, e.g., an expression vector containing apolynucleotide fragment encoding said peptide, where the polynucleotidefragment is operably linked to a promoter capable of expressing mRNAfrom the fragment in the host cell.

In another aspect, the invention includes a method of reducing ischemicinjury to a cell, tissue or whole organ exposed to hypoxic conditions.The method includes introducing into the cell, tissue or whole organprior to exposure to hypoxic conditions, a therapeutically-effectiveamount of an isozyme-specific δPKC antagonist, such as δV1-1, δV1-2,δV1-5, or any of the modification, derivatives, and fragments of thesepeptides described above. The δPKC antagonist inhibits δPKC, resultingin protection of the cell, tissue or whole organ by reducing ischemicinjury to the cell. The reduction of ischemic injury is measuredrelative to the ischemic injury suffered by a corresponding cell, tissueor whole organ that did not undergo δPKC antagonist peptidepretreatment.

It will be appreciated that the dose of peptide administered will varydepending on the condition of the subject, the timing of administration(that is, whether the peptide is administered prior to, during, or afteran ischemic event). Those of skill in the art are able to determineappropriate dosages, using, for example, the dosages used in the wholeorgan and animal studies described herein.

The method can be practiced with a variety of cell types, includingcardiac cells, central nervous system (CNS) cells (e.g., neurons, glialcells), kidney cells and the like. A variety of tissue or whole organscan be treated, including but not limited to the brain, heart, eye, andkidney.

The peptides can be administered to the cell, tissue or whole organ invitro, in vivo, or ex vivo. All modes of administration arecontemplated, including intravenous, parenteral, subcutaneous,inhalation, intranasal, sublingual, mucosal, and transdermal. Apreferred mode of administration is by infusion or reperfusion througharteries to a target organ, such as through the coronary arteries to anintact heart.

In yet another aspect, the invention includes a method of enhancingischemic injury to a cell, tissue or whole organ exposed to hypoxicconditions. This method is relevant to, for example, the treatment ofsolid tumors in subjects. The method also finds use in in vitro or invivo research where damage to a cell or tissue is desired. The methodincludes introducing into the cell, tissue or whole organ atherapeutically-effective amount of an isozyme-specific δPKC agonist,such as ψδRACK (SEQ ID NO:6) or any of the peptides obtained from amodification to ψδRACK as discussed above. The extent of enhancedischemic injury is measured relative to the ischemic injury suffered bya corresponding cell, tissue or whole organ untreated with a δPKCagonist.

IV. Identification and Screening of Test Compounds

In another aspect, the invention includes methods of identifyingcompounds effective to induce protection of a cell or tissue fromhypoxic/ischemic damage or to enhance hypoxic or ischemic damage in acell or tissue.

In the first method, the δPKC-specific agonists δV1-1, δV1-2, δV1-5 orany of the modifications of these peptides described above, are used toidentify compounds effective to inhibit δPKC translocation in cellsand/or to competitively displace the peptide from Annexin V (SEQ IDNO:72) or other δRACK and/or to prevent or inhibit the peptide frombinding to such a δRACK. Such compounds find use as therapeutic agentsto inhibit δPKC translocation and/or function to thereby induceprotection of cells or tissues from damage due to ischemia. Thecompounds also find use as screening tools to identify other peptides orcompounds suitable for the same purpose.

In this method, a δPKC peptide containing a δRACK binding site, such asAnnexin V, is brought into contact with a δPKC antagonist peptide withthe δRACK binding site, such as δV1-1, δV1-2, δV1-5, in the presence andabsence of a test compound. The interaction of the test compound withthe peptide having the δRACK binding site is monitored and/or thecatalytic activity of the δPKC agonist or the test compound ismonitored. Generally, the test compound is identified as being effectiveto induce protection from an ischemic or an hypoxic event if, in thepresence of the test compound, binding of the peptide antagonist to theδRACK binding site is decreased, relative to binding in the absence ofthe test compound. Alternatively, the catalytic activity of thecomponents can be monitored. For example, the phosphorylation activityof the peptides can be monitored. If the ability of the test compound toincrease phosphorylation, or some other catalytic activity subsequent tobinding, is increased relative to activity in the absence of the testcompound then the compound is identified as being effective to induceprotection from damage caused by either a hypoxic or an ischemic event.

In another method, the agonist peptide ψδRACK can be used to identifycompounds effective to enhance hypoxic or ischemic damage in a cell ortissue. In this method, a ψδRACK agonist peptide is brought into contactwith a δPKC peptide containing a δRACK binding site in the presence andabsence of a test compound. The test compound, if able to decreasebinding of the peptide agonist to the δRACK binding site relative tobinding in the absence of the test compound, is identified as beingeffective to enhance damage due to ischemia. Suitable ψδRACK peptidesinclude the peptide identified as SEQ ID NO:6, fragments, andderivatives thereof, including but not limited to those set forth in SEQID NOS:10-24.

ψδRACK-like compounds can also be identified by measuring its effect onthe catalytic activity of δPKC in vitro. The desired compound willincrease the catalytic activity of δPKC in the presence of limitingamounts of δPKC co-factors (Ron et al., 1995). Catalytic activity refersto the ability of the peptide to phosphorylate another protein orsubstrate.

Experimental details of a similar screening method are set forth in U.S.Pat. No. 6,165,977, and this portion on Col. 14, line 45-Col 15, line 54is incorporated by reference herein. In brief, and by way of example foridentifying a compound effective to protect a cell or tissue fromischemia, δPKC is immobilized inside the wells of a multiwell plate byintroducing a solution containing δPKC into the plate and allowing theδPKC to bind to the plastic. The wells may be precoated with substancesthat enhance attachment of δPKC and/or that decrease the level ofnon-specific binding.

The plate is then incubated with a blocking solution (containing, forexample bovine serum albumin) and then washed several times. A solutioncontaining reporter-labelled (e.g., radiolabelled orfluorescently-tagged) peptide δV1-1 (SEQ ID NO: 4) and, in the testwells, as opposed to the control wells, a test compound is added.Different wells may contain different test compounds or differentconcentrations of the same test compound. Each test compound at eachconcentration is typically run in duplicate and each assay is typicallyrun with negative (wells with no test compound) as well as positive(wells where the “test compound” is unlabeled peptide) controls. Thefree peptide is then washed out, and the degree of binding in the wellsis assessed.

A test compound is identified as active it if decreases the binding ofthe peptide, i.e., its effect on the extent of binding is above athreshold level. More specifically, if the decrease in binding is aseveral-fold different between the control and experimental samples, thecompound would be considered as having binding activity. Typically, a2-fold or 4-fold threshold difference in binding between the test andcontrol samples is sought.

Detection methods useful in such assays include antibody-based methods,direct detection of a reporter moiety incorporated into the peptide,such as a fluorescent label, and the like.

A variety of test compounds may be screened, including other peptides,macromolecules, small molecules, chemical and/or biological mixtures,fungal extracts, bacterial extracts or algal extracts. The compounds canbe biological or synthetic in origin.

From the foregoing, it can be seen how various objects and features ofthe invention are met. New activator and inhibitor peptides of δPKCtranslocation and function were identified. The peptides can bedelivered in vivo or ex vivo to achieve a functional inhibition oractivation of δPKC. For example, delivery of the peptides to an intactheart via the coronary artery permits the peptides to act as a directpeptide modulator of protein-protein interactions intracellularly. Itwas also found that inhibition of δPKC by delivery of a δPKC antagonistreduces tissue damage due to an ischemic event. It is noteworthy thatδPKC and εPKC (previously described in the art) exhibit an opposingeffect in response to ischemia, yet activation of both isozymes leads toa similar form of cardiac hypertrophy. This was particularly unexpected,because both isozymes are activated by ischemia as well as by stimulithat lead to cardioprotection from ischemia (Gray, M. O. et al., Chen,C.-H, et al.) δPKC and εPKC are opposing forces and a balance betweenthese opposing forces likely determines the outcome to the ischemicinsult, where protection occurs when activation of εPKC exceeds that ofδPKC. During a long ischemic period, there may be an advantage to inducecell death, which will result from a time-dependent increase in theactivity of δPKC relative to that of εPKC. In that way, the limitedamounts of oxygen, glucose and other nutrients could be used by theremaining, less damaged, cells, ultimately leading to an improvedoutcome to the organ.

V. Examples

The following examples further illustrate the invention described hereinand are in no way intended to limit the scope of the invention.

Example 1 Activity of δV1-1, δV1-2 and ψδRACK

A. Peptide Preparation

δV1-1 (SEQ ID NO:4) and ψδRACK (SEQ ID NO:6) were synthesized andpurified (>95%) at the Stanford Protein and Nucleic Acid Facility. Thepeptides were modified with a carrier peptide by cross-linking via anN-terminal Cys-Cys bond to the Drosophila Antennapedia homeodomain (SEA)ID NO:8; Théodore, L., et al; Johnson, J. A. et al., 1996b). In somestudies not reported here, the peptides were lined to Tat-derivedpeptide (SEQ ID NO:9).

B. Peptide Delivery Into Cells

Primary cardiac myocyte cell cultures (90-95% pure) were prepared fromnewborn rats (Gray. M. O. et al.; Disatnik M.-H. et al.). The peptidesδV1-1 and ψδRACK were introduced into cells at an applied concentrationof 500 nM in the presence and absence of phorbol 12-myristate 13-acetate(PMA) at concentrations of 3 nm and 10 nm, respectively, for 10-20minutes. In a third set of cells, the peptide δV1-1 was applied at aconcentration of 500 nM in the presence and absence of 500 nM ψδRACK.

Translocation of δPKC isozyme was assessed by using δPKCisozyme-specific antibodies in Western blot analysis (Santa CruzBiotechnology). Western blot analysis of cystosolic and particulatefractions of treated cells was carried out as described by Johnson etal., 1995. Subcellular localization of δPKC isozymes was assessed bychemiluminescence of blots probed with anti-δPKC, anti-αPKC andanti-εPKC antibodies. Amounts of PKC isozymes in each fraction wasquantitated using a scanner and translocation is expressed as the amountof isozymes in the particulate fraction over the amount of isozymes innon-treated cells. Changes in translocation of δPKC isozyme were alsodetermined by immunofluorescence study of treated and fixed cells (Grayet al., 1997). Translocation was determined by counting over 100cells/treatment in a blinded fashion. The results are shown in FIG.2A-2B, FIGS. 3A-3B and FIGS. 4A-4B.

Example 2 Peptide Administration to Isolated Cardiac Myocytes

The peptides δV1-1 and ψδRACK were prepared as described in Example 1.

Adult male Wistar rat cardiomyocytes were prepared on a Langendorffapparatus (van der Heide, R. S. et al., J. Mol. Cardiol., 22:165 (1990))by collagenase treatment (Armstrong, S. et al., Cardiovasc. Res., 28:72(1994)). The cells were treated with δV1-1 at concentrations of 10 nM,100 nM, 500 nM, and 1 μM in the presence or absence of 1 μM ψδRACK.βPKC-selective activator was used as a control.

For stimulated ischemia, adult myocytes treated in microcentrifuge tubeswith δV1-1 and/or ψδRACK peptides conjugated to the carrier were washedtwice with degassed glucose-free incubation buffer and pelleted. On topof a minimal amount of buffer, the cell pellets were overlaid witheither a micro-balloon (Sig Manufacturing, Montezuma, Iowa) or withdegassed buffer saturated with nitrogen, and sealed with an airtighttop. Tubes were then incubated at 37° C. for either 180 minutes or 90minutes.

Cell damage was assessed by an osmotic fragility test by measuring theuptake of trypan blue added in a hypotonic (85 mosM) solution. Theresults are shown in FIGS. 5A-5B. Similar results were also obtained byusing a live-dead kit (Molecular Probes) or measuring the release oflactose dehydrogenase to the medium using a kit (Sigma) as previouslydescribed (Chen, et al., 1999; Gray et al., 1997; Mackay, K., et al. J.Biol. Chem., 221:6272-6279 (1999)).

Example 3 Ex Vivo Peptide Administration to Whole Hearts and Effect onCell Damage

Adult, male rats were anesthetized with i.p. avertin, and their heartswere rapidly removed and cannulated via the aorta for perfusion asdescribed in the art (Colbert, M. C. et al, J. Clin. Invest., 100:1958(1997)) using Langendorff set-up. Care was taken to have the heartsperfused within 90 seconds of removal. The hearts were perfused withoxygenated Krebs-Henseleit solution comprised of, in nmol/L, NaCl 120;KCl 5.8; NaHCO₃ 25; NaH₂O₄ 1.2; MgSO₄ 1.2; CaCl₂ 1.0; and dextrose 10,pH 7.4 at 37° C.

After a 10-20 minute equilibration period, the hearts were perfused withδV1-1 peptide (SEQ ID NO:4) or with ψδRACK peptide (SEQ ID NO:6),prepared as described in Example but conjugated to a Tat-derived peptide(Tat 47-57, SEQ ID NO:9), for 20 minutes. Perfusion was maintained at aconstant flow of 10 mL/min with Krebs-Hanseleit solution containing 0.5μM of the appropriate peptide. The Langendorff method employed usedretrograde flow from the ventricle to the aorta and into the coronaryarteries, bypassing the pulmonary arteries.

To induce global ischemia, flow was interrupted for 30 minutes. Afterthe ischemic event, the hearts were re-perfused for 30-60 minutes.During reperfusion, ischemia-induced cell damage was determined bymeasuring the activity of creatine phosphokinase (CPK) (absorbance at520 nm) in the perfusate using a Sigma kit. As controls, some ex vivohearts were left untreated, or maintained under normoxia conditions, ortreated with the Tat-carrier peptide alone, or treated with Tat-carrierpeptide conjugated to a scrambled δV1-1 peptide. The results are shownin FIGS. 6A-6B.

Example 4 Ex Vivo Peptide Administration to Whole Hearts and Effect onFunctional Recovery

Rat hearts were isolated as described in Example 3. The left ventricularpressure and its real-time derivative (dP/dt) were monitored via a latexballoon placed in the ventricular cavity and at a constant heart rate bypacing (3.3 Hz) and at a constant coronary flow (10 ml/min.). The heartswere subjected to 20 minutes of ischemia and 30 minutes of reperfusion.During the first 20 minutes of reperfusion, 500 nM of δV1-1 or vehiclecontrol was delivered. The results are shown in FIGS. 7A-7B.

Example 5 In Vivo Administration of δV1-1 After Ischemia

Adult female pigs, 35-40 kg in weight, were anesthetized and a catheterwas introduced through the carotid artery into the heart. Usingconventional intervention cardiology techniques, a wire was placedthrough a catheter and into the left anterior descending artery. Aballoon was run over this wire to a site of occlusion where it was theninflated to block blood flow for 30 minutes. During the last 10 minutesof the 30-minute occlusion, either a control comprised of the carrierpeptide alone or δV1-1 peptide (conjugated to a carrier Tat peptide asdescribed in Example 3 was delivered by slow diffusion (1 mL/min)directly downstream of the occlusion. Approximately 20 μg of δV1-1peptide (˜400 ng per kg body weight) was administered.

After 30 minutes of occlusion, the balloon was removed and pigs wereleft to recover from surgery. Five days later, the pigs were euthanizedand hearts were harvested. After heart removal, the LAD was occluded.With the occlusion in place, Evans Blue dye, which stains all areas notat risk of infarct in blue while leaving all areas with no access toblood flow red, was infused. Hearts were then cut into slices andstained with a tetrazolium red dye which stains all live areas red andinfracted dead tissue in white. Each heart had multiple tissue sliceswith distinctive areas marking the area at risk for ischemia and theinfracted area. From this the percent infarct per area at risk for eachslice and for the entire heart was determined. The results are shown inFIGS. 9A-9C.

Example 6 Administration of δV1-1 to Rats for Stroke Damage Protection

A. Cerebral Ischemia Model

Adult male Sprague-Dawley rats weighing between 280-320 g were used.Animals were maintained under isofluorane anesthesia during all surgicalprocedures. Physiological parameters were monitored and maintained inthe normal range. Rectal temperature was also measured. At thecompletion of the experiment, the animals were euthanized with abarbiturate overdose and prepared for histological analysis.

B. Focal Model

Ischemia was induced using an occluding intraluminal suture. An uncoated30 mm long segment of 3-0 nylon monofilament suture with the tip roundedby flame was inserted into the stump of the common carotid artery andadvanced into the internal carotid artery approximately 19-20 mm fromthe bifurcation in order to occlude the ostium of the middle cerebralartery. Sham control animals underwent similar anesthesia and surgicalmanipulation, but did not experience ischemia. At the end of a 2 hourischemic period, the suture was removed and the animal allowed torecover. Brains were harvested after 24 hrs of reperfusion.

C. Peptide Delivery

δV1-1 (SEQ ID NO:4) conjugated to Tat peptide (0.05 mL, SEQ ID NO:8) orTat carrier control peptide (50 μL of 10 μM solution in saline) wereinjected into the carotid artery either immediately before or before andafter the 2 hours occlusion. The final blood concentration of δV1-1 was1 μM.

D. Histology

Animals were perfused with heparinized saline and brains removed andsectioned into 2 mm thick slices. To assess ischemic injury, brainsections were stained with cresyl violet or with triphenyl tetrazoliumchloride, a live tissue stain to indicate the regions of infarct. Areasof infarction (white) were then measured using an image analysis systempreviously described (Yenari, M. A. et al., Brain Res., 739:36-45(1998).; Maier, C. et al., Stroke, 29:2171-2180 (1998).). The resultsare shown in FIGS. 11A-11B.

Although the invention has been described with respect to particularembodiments, it will be apparent to those skilled in the art thatvarious changes and modifications can be made without departing from theinvention.

It is claimed:
 1. A composition comprising a synthetic peptideconsisting of δV1-2 (SEQ ID NO:5) linked to a moiety effective tofacilitate transport across a cell membrane, wherein the syntheticpeptide is at least 95% of the composition.
 2. The composition of claim1, wherein the moiety is selected from the group consisting of aTat-derived peptide, an Antennapedia carrier peptide, and a polyargininepeptide.
 3. The composition of claim 1, wherein the peptide and themoiety are linked to form a fusion peptide.
 4. The composition of claim3, wherein the moiety is selected from the group consisting of aTat-derived peptide, an Antennapedia carrier peptide, and a polyargininepeptide.
 5. The composition of claim 3, wherein the moiety comprises anamino acid sequence selected from the group consisting of SEQ ID NO:8and SEQ ID NO:9.
 6. The composition of claim 3, wherein the moietyconsists of an amino acid sequence selected from the group consisting ofSEQ ID NO:8 and SEQ ID NO:9.
 7. The composition of claim 3, wherein themoiety comprises the amino acid sequence of SEQ ID NO:9.
 8. Thecomposition of claim 3, wherein the moiety consists of the amino acidsequence of SEQ ID NO:9.
 9. The composition of claim 1, wherein thepeptide and the moiety are linked by a Cys-Cys bond.
 10. The compositionof claim 9, wherein the moiety is selected from the group consisting ofa Tat-derived peptide, an Antennapedia carrier peptide, and apolyarginine peptide.
 11. The composition of claim 9, wherein the moietycomprises an amino acid sequence selected from the group consisting ofSEQ ID NO:8 and SEQ ID NO:9.
 12. The composition of claim 9, wherein themoiety consists of an amino acid sequence selected from the groupconsisting of SEQ ID NO:8 and SEQ ID NO:9.
 13. The composition of claim9, wherein the moiety comprises the amino acid sequence of SEQ ID NO:9.14. The composition of claim 9, wherein the moiety consists of the aminoacid sequence of SEQ ID NO:9.
 15. A conjugate comprising: a firstpeptide consisting of δV1-2 (SEQ ID NO:5) and a terminal cysteine, and asecond peptide consisting of the amino acid sequence of SEQ ID NO:9,wherein the first and second peptides are linked by a Cys-Cys bond.